An animal's immune system provides a stream of protein molecules know as antibodies that circulate through the animal's blood stream. When large toxin molecules or microorganisms, known as antigens, enter the blood stream, the immune system, through a complex biochemical sequence, recognizes that the antigens are foreign to the animal's system, and hence, a threat. In response to the invasion of antigens, the immune system rapidly produces “customized” antibodies that attach themselves to the specific site on the antigen. The attached antibodies act as markers to identify the antigens or other material produced by the immune system, which in turn destroy the antigens.
Because antibodies are extremely specific; selective; and sensitive, and relatively easy to generate, scientists have recognized that they can be the basis for a variety of useful tests known as immunoassays. In its basic form, an immunoassay for a specific antigen involves allowing a test sample of material of unknown composition, an analyte, to come in contact with immobilized antibodies that bind to the specific antigen. If the antigen is present, it will bind, i.e. conjugate, with the antibodies and also become immobilized. The immobilized antibodies are washed to remove any free analyte, and then treated with labeled antibodies that bind to a different site on the antigen than the immobilized antibodies. If the antigen in question was present in the analyte, it will now be conjugated to the labeled antibodies and also to the immobilized antibodies. Presence of the antigen can be detected by sensing the label.
Typically, labels have distinct signatures detectable by electromagnetic radiation absorption, emission, or both. A particularly useful technique is to use labels that are florescent. That is, they absorb electromagnetic radiation above the frequency range of visible light then instantly emit visible light. The strength of the absorption or emission can be directly correlated to the amount of label, and hence the antibody being observed. Alternatively, a label detectable by electronic means may be used. For example, the label may impede a radio frequency signal and the amount of impedance detected by electronic means.
Thus, Immunoassays are tests that take advantage of the specific binding of an antibody to its antigen and are discussed and illustrated in most University level biochemistry textbooks. For example, see L. Nelson, et al. Lehninger Principles of Biochemistry, 3rd Ed., 231-233, Worth, N.Y. (2000) and L. Stryer, Biochemistry, 4th Ed., 60-63, W.H. Freeman, NY (1995). The main characteristic of immunological techniques is the appropriate labeling of the antibody or the antigen. This label helps create a signal that correlates with the immunoreaction and allows the detection of the analyte of interest. In laboratory assays, various wash steps are required to remove free labeled or unlabeled reactants and allow the detection of the analyte by the bound and labeled reactants. Test results may be quantified by comparison to a calibration curve established by a previously run series of assays using known amounts of analyte.
A widely practiced form of immunoassay is the enzyme-linked immunosorbent assay (ELISA); supra, Nelson, et al., and Stryer. There are many variations of ELISA's, most of which require multiple steps and a moderate to extensive skill level to execute. However, one category of immunoassay, i.e. the well-known lateral flow immunoassay (LFIA), of which home pregnancy tests are an example, are simple, typically requiring only one step, and requiring no technical sophistication to perform. Thus, LFIA can be easily performed by non-trained users and used on-site during sample collection. The simplicity of the tests paired with their quick return of results (2-15 minutes), means that testing is cost-effective. LFIA represent an appropriate point-of-care (POC) and field-use technology that can be applied to a broad range of applications. Despite the advantages of LFIA, they are often limited to simple screening applications. This is because LFIA's, in their present form, are not easily quantifiable and are not sensitive enough for certain applications.
Each year millions of patients, about a third of those hospitalized, are exposed to heparin. About 1% to 5% of these heparin-exposed patients develop a severe complication known as heparin-induced thrombocytopenia often referred to as “HIT”. Venous or arterial thrombosis is among the effects of HIT, and in patients suffering from acute thrombosis, HIT may be fatal. After discontinuation of heparin in patients with HIT, the platelet levels generally return to normal. Therefore, timely and accurate diagnosis of HIT can alleviate pain and even prevent death. See Arapally et al, N Engl J Med, 355; 8: 809.
The immunoglobulin antibodies, such as IgG, IgA, IgE, or IgM antibodies, that develop after five or more days of heparin therapy appear to cause HIT. These antibodies differ from those associated with other forms of drug-induced thrombocytopenia in that, in the presence of optimal concentrations of heparin, they activate blood platelets. This activation causes the platelets to release the contents of their storage granules and to undergo membrane changes that create sites for the binding of a coagulation factor, fibrinogen, normally present in plasma (B. H. Chong, et al., Br. J. Haematol., 64: 347 (1986)). Heparin first binds to platelet factor 4 (PF4), which arises during heparin treatment, to form a highly immunogenic complex on the surface of platelets. Next, in susceptible patients, immunoglobulin, e.g. IgG, IgA, IgE, or IgM antibodies to the antigenic heparin-PF4 complex develop that bind with the complex to activate platelets via Fc receptors on the surface of the platelets (M. F. Cooney, Critical Care Nurse, 26, 6: 30 (2006).
Several HIT diagnostic procedures and assays are reported in the art, but each has drawbacks that limits its use in accurately, rapidly, reliably, and cost effectively diagnosing the risk of HIT. For example, U.S. Pat. No. 5,972,718 teaches an ELISA type immunoassay. However, while ELISA procedures and assays are suited for laboratory environments, they are not well suited for POC use because of their complexity and the requirement for skilled operators. In practice, an ELISA requires 3-4 hours of skilled technician time and typically involves turnaround times of one day to one week.